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TRAPP stimulates guanine nucleotide exchange on Ypt1p.

Wang W, Sacher M, Ferro-Novick S - J. Cell Biol. (2000)

Bottom Line: Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p.Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p.Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and the Department of Cell Biology Yale University School of Medicine, New Haven, Connecticut 06519-1418, USA.

ABSTRACT
TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5. Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP. Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p. Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p. Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p. Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP.

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[3H]GDP release from Ypt1p in the presence of cell lysates prepared from ts mutants that are defective in different TRAPP subunits. His6-Ypt1p preloaded with [3H]GDP was incubated with cell lysates for 10 min at 30°C (A) or 37°C (B) as described in the Materials and Methods. Lysates assayed at 37°C were pre-incubated for 5 min at 37°C immediately before the reaction was performed. The average intrinsic rate of [3H]GDP release from Ypt1p was measured in the presence of BSA (4 mg/ml) and the value obtained (15.5 ± 2% at 30°C; 28.0 ± 2% at 37°C) was subtracted as background. The numbers reported are the average of two separate experiments.
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Figure 5: [3H]GDP release from Ypt1p in the presence of cell lysates prepared from ts mutants that are defective in different TRAPP subunits. His6-Ypt1p preloaded with [3H]GDP was incubated with cell lysates for 10 min at 30°C (A) or 37°C (B) as described in the Materials and Methods. Lysates assayed at 37°C were pre-incubated for 5 min at 37°C immediately before the reaction was performed. The average intrinsic rate of [3H]GDP release from Ypt1p was measured in the presence of BSA (4 mg/ml) and the value obtained (15.5 ± 2% at 30°C; 28.0 ± 2% at 37°C) was subtracted as background. The numbers reported are the average of two separate experiments.

Mentions: Wild-type and ts mutant extracts were incubated with Ypt1p that was preloaded with [3H]GDP and assayed for release activity at 30° and 37°C as described above. As was found for bet3-1, extracts prepared from the bet5-1, trs31-1, and trs130ts2 mutants were partially defective for activity at 30°C (Fig. 5 A) and more dramatically affected at 37°C (Fig. 5 B). As a control, we also assayed a lysate prepared from trs85Δ. Whereas BET3, BET5, TRS31, and TRS130 are essential for the vegetative growth of yeast, TRS85 is dispensable (Sacher et al. 2000). As anticipated, the absence of Trs85p did not lead to a loss of activity at either 30°C (Fig. 5 A) or 37°C (Fig. 5 B).


TRAPP stimulates guanine nucleotide exchange on Ypt1p.

Wang W, Sacher M, Ferro-Novick S - J. Cell Biol. (2000)

[3H]GDP release from Ypt1p in the presence of cell lysates prepared from ts mutants that are defective in different TRAPP subunits. His6-Ypt1p preloaded with [3H]GDP was incubated with cell lysates for 10 min at 30°C (A) or 37°C (B) as described in the Materials and Methods. Lysates assayed at 37°C were pre-incubated for 5 min at 37°C immediately before the reaction was performed. The average intrinsic rate of [3H]GDP release from Ypt1p was measured in the presence of BSA (4 mg/ml) and the value obtained (15.5 ± 2% at 30°C; 28.0 ± 2% at 37°C) was subtracted as background. The numbers reported are the average of two separate experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2192651&req=5

Figure 5: [3H]GDP release from Ypt1p in the presence of cell lysates prepared from ts mutants that are defective in different TRAPP subunits. His6-Ypt1p preloaded with [3H]GDP was incubated with cell lysates for 10 min at 30°C (A) or 37°C (B) as described in the Materials and Methods. Lysates assayed at 37°C were pre-incubated for 5 min at 37°C immediately before the reaction was performed. The average intrinsic rate of [3H]GDP release from Ypt1p was measured in the presence of BSA (4 mg/ml) and the value obtained (15.5 ± 2% at 30°C; 28.0 ± 2% at 37°C) was subtracted as background. The numbers reported are the average of two separate experiments.
Mentions: Wild-type and ts mutant extracts were incubated with Ypt1p that was preloaded with [3H]GDP and assayed for release activity at 30° and 37°C as described above. As was found for bet3-1, extracts prepared from the bet5-1, trs31-1, and trs130ts2 mutants were partially defective for activity at 30°C (Fig. 5 A) and more dramatically affected at 37°C (Fig. 5 B). As a control, we also assayed a lysate prepared from trs85Δ. Whereas BET3, BET5, TRS31, and TRS130 are essential for the vegetative growth of yeast, TRS85 is dispensable (Sacher et al. 2000). As anticipated, the absence of Trs85p did not lead to a loss of activity at either 30°C (Fig. 5 A) or 37°C (Fig. 5 B).

Bottom Line: Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p.Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p.Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and the Department of Cell Biology Yale University School of Medicine, New Haven, Connecticut 06519-1418, USA.

ABSTRACT
TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5. Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP. Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p. Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p. Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p. Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP.

Show MeSH
Related in: MedlinePlus